1. Field of the Invention
The present invention relates to a general method for isolation and growth of nitrite-resistant bacteria with a high level of inducible nitrate reductase activity. In particular, the present invention relates to a method of isolation and growth of bacteria with a high level of inducible nitrate reductase activity using Chinese cabbage and celery extracts as a source of the bacteria and media.
The present invention also relates to a general method of inhibiting bacterial growth in foods by adding nitrate-rich vegetable extracts. More specifically, the present invention demonstrates an anti-bacterial effect of Chinese cabbage and celery extracts on beef during storage.
2. Description of Related Art
Sodium nitrate and nitrite are added to meat as preservatives to ward off bacteria. These compounds are also responsible for a bright-red color of cured meat (Committee on nitrite and alternative curing agents in food, national Academy of Sciences, Chapter 3, 1981). Vegetables such as celery, spinach, beets, turnip greens, radishes and lettuce have a high nitrate content (Committee on nitrite and alternative curing agents in food, national Academy of Sciences, Chapter 5, 1981). As a result, nitrate consumed by U.S. residents is primarily through vegetables. However, the main source of nitrite in the U.S. is cured meats (Committee on nitrite and alternative curing agents in food, national Academy of Sciences, Chapter 5, 1981; White, 1975).
In mammalian cells, nitric oxide synthase catalyzes formation of nitric oxide from L-arginine. Nitric oxide plays a significant role in signal transduction and cell to cell communication. Nitric oxide release can be assayed by measuring its stable degradation products, nitrite and nitrate (Schmidt, 1995).
The most commonly used method for analysis of nitrite is a colorimetric method using the Griess reaction (Committee on nitrite and alternative curing agents in food, national Academy of Sciences, Chapter 5, 1981; Schmidt, 1995). Nitrate is determined by measurement of nitrite by the Griess reaction after reduction of nitrate to nitrite (Committee on nitrite and alternative curing agents in food, national Academy of Sciences, Chapter 5, 1981; Schmidt, 1995). Nitrate is reduced to nitrite by passing the reaction mixture through a column packed with cadmium shavings or by adding granulated cadmium in the mixture (Committee on nitrite and alternative curing agents in food, national Academy of Sciences, Chapter 5, 1981; Vodovotz, 1996).
Recently, nitrate was enzymatically reduced to nitrite by incubation with nitrate reductase. Nitrate reductase has been studied in bacteria, fungus and plants. Thus far three different kinds of nitrate reductases have been found. Assimilatory nitrate reductase converts nitrate to ammonia, vianitrite, which is assimilated to nitrogen metabolism.
Ammonia, the end product of the pathway inhibits nitrate reductase activity and oxygen does not have any effect on the nitrate reductase activity (Kubo, et al., 1988; Hyde et al., 1990). A membrane-bound, respiratory (dissimilatory) nitrate reductase converts nitrate to nitrite (Carlson, et al. 1982) and then the formed nitrite is converted to nitric oxide and nitrous oxide by respiratory nitrite reductase (Smith, et al. 1992). This pathway, which produces ATP from ADP using nitrate in anaerobic condition, is insensitive with ammonia.
Recently, the third nitrate reductase, periplasmic nitrate reductase, has been found. Contrary to the other nitrate reductases, the periplasmic nitrate is not encoded on the chromosome, but by the megaplasmid and the gene products with a signal peptide are found in the periplasm (Bursakov, et al. 1997; Siddiqui, et al. 1993; Carter, et al. 1995). This nitrate reductase may be involved in cellular redox balance.
A few commercially available nitrate reductases are nitrate reductase which are obtained from E. coli (Worthington Biochemical Co. and Sigma Co, Aspergillus (Boehringer-Mannheim), and corn (The Nitrate Elimination Co.).
The significance of the presence of nitrite and nitrate in meat relates to health concerns. Adding nitrite and nitrate to meat became a health problem because sometimes nitrite reacts with the amines in meat. This reaction forms a chemical, nitrosamines, that is found to cause cancer in laboratory animals (Graham, 1980). To avoid the health problem caused by this nitrosamine formation, some meat products contain BHA and BHT, preservatives of another kind. However, neither of these preservatives are naturally occurring.
It would therefore be useful to develop a method for the isolation and growth of nitrite released in bacteria with high nitrate reductase activity. Additionally, it would be useful to develop a method for the retardation of bacterial growth in foods using plant extracts.